Pleiotrophin, a paracrine regulator of hematopoietic stem cell fate

描述（由申请人提供）：最近的研究表明，骨髓（BM）中造血干细胞（HSC）的维持和保留依赖于来自BM微环境或龛内细胞的外源性信号，包括血管内皮细胞（EC）、血管周围细胞、骨系细胞、间充质基质细胞和交感神经元。然而，小生境细胞单独或协同调节HSC稳态的确切机制尚未完全阐明。此外，造血干细胞再生的控制机制，这是必要的骨髓抑制化疗和放疗后的造血系统的恢复，仍然不太清楚。在这里，我们将使用创新的小鼠模型和分子技术来定义的机制，通过该机制，新的分泌蛋白，多效营养因子（PTN），在体内调节HSC的命运，并确定BM小生境细胞负责PTN介导的维持和再生的HSC池。HSC支持EC的差异基因表达分析显示PTN的过度表达，PTN是一种肝素结合生长因子，通常在海马中表达，并作为神经突生长因子发挥作用。用PTN短期培养小鼠BM HSC导致长期再增殖的HSC扩增超过10倍，而BM微环境中PTN的缺失也导致小鼠HSC含量的10倍缺陷。解剖学研究表明，PTN由BM中的VEGFR 3 + VE钙粘蛋白+窦状内皮细胞和CXCL 12+血管周围细胞表达，表明PTN是HSC血管生态位的独特组分。在初步研究中，PTN介导的造血干/祖细胞体外扩增被Ras或MEK的药理学抑制剂废除，表明PTN通过诱导Ras/MEK信号传导途径介导HSC生长。基于这些发现，我们假设PTN是骨髓血管龛内HSC库的旁分泌调节剂，并通过激活Ras/MEK/ERK通路调节HSC生长。根据这一假设，我们提出了以下具体目的：1）确定Ras/MEK/ERK途径在介导PTN对HSC的作用中的作用，2）确定哪些HSC小生境细胞负责介导PTN对HSC维持和再生的体内作用，和3）确定PTN是否可以通过激活Ras/MEK信号传导途径离体或体内扩增人HSC。我们广泛的长期目标是确定PTN介导HSC自我更新和再生的机制，以促进PTN的翻译，用于人类HSC的治疗性扩增。

DESCRIPTION (provided by applicant): Recent studies have revealed that the maintenance and retention of hematopoietic stem cells (HSCs) in the bone marrow (BM) are dependent upon extrinsic signals from cells within the BM microenvironment or niche, including vascular endothelial cells (ECs), perivascular cells, osteolineage cells, mesenchymal stromal cells and sympathetic neurons. However, the precise mechanisms through which niche cells individually or in concert regulate HSC homeostasis have yet to be fully elucidated. Furthermore, the mechanisms controlling HSC regeneration, which is necessary for the recovery of the hematopoietic system following myelosuppressive chemo- and radiotherapy, remain less well understood. Here, we will use innovative mouse models and molecular techniques to define the mechanism through which the novel secreted protein, pleiotrophin (PTN), regulates HSC fate in vivo and to identify the BM niche cells responsible for PTN-mediated maintenance and regeneration of the HSC pool. Differential gene expression analysis of HSC-supportive ECs revealed the overexpression of PTN, a heparin binding growth factor, which is normally expressed in the hippocampus and functions as a neurite outgrowth factor. Short-term culture of murine BM HSCs with PTN caused more than 10-fold expansion of long-term repopulating HSCs, whereas deletion of PTN in the BM microenvironment also caused a 10-fold deficit in HSC content in mice. Anatomic studies suggest that PTN is expressed by VEGFR3+VEcadherin+ sinusoidal ECs and CXCL12+ perivascular cells in the BM, suggesting that PTN is a unique component of the HSC vascular niche. In preliminary studies, PTN-mediated expansion of hematopoietic stem/progenitor cells in vitro was abrogated by pharmacologic inhibitors of Ras or MEK, suggesting that PTN mediates HSC growth via induction of the Ras/MEK signaling pathway. Based upon these findings, we hypothesize that PTN is a paracrine regulator of the HSC pool within the BM vascular niche and regulates HSC growth via activation of the Ras/MEK/ERK pathway. In keeping with this hypothesis, we propose the following Specific Aims: I) Determine the role of the Ras/MEK/ERK pathway in mediating PTN effects on HSCs, 2) Determine which HSC niche cells are responsible for mediating PTN effects on HSC maintenance and regeneration in vivo, and 3) Determine whether PTN can expand human HSCs ex vivo or in vivo via activation of Ras/MEK signaling pathway. Our broad, long-term objective will be to define the mechanisms through which PTN mediates the self-renewal and regeneration of HSCs to facilitate translation of PTN for the therapeutic expansion of human HSCs.